Petroleum serves as the primary source of automotive fuel and as the dominating feedstock for organic chemicals and plastics. The finite nature of the petroleum reserves and the negative environmental impact of petroleum use have shifted attention towards alternatives from renewable feedstocks (1-4). Fermentation of carbohydrates has been shown to produce short chain hydroxy acids as well as other chemicals that can be polymerized into plastics and replace petroleum (5). Commercial production of lactic acid using pure bacterial cultures started as early as 1895 (6) and current annual production is >300,000 metric tons. Although lactic acid is primarily used by food and pharmaceutical industries, its application in production of polylactic acid biopolymers (PLA) is expected to exceed other uses, provided the cost of PLA production can be lowered (7, 8).
Lactic acid is condensed into lactide dimers, purified and polymerized into a thermoplastic (7, 9). Blending D(−)- and L(+)-lactic acid polymers provides substantial control of the physical and thermochemical properties and the rate of biodegradation (9). Although lactic acid can be synthesized from petroleum, chemical synthesis produces a mixture of isomers that are not suitable for PLA. Optically pure lactic acid required for PLA synthesis can be readily produced by microbial fermentation (8). L(+)-lactic acid is produced commercially by lactic acid bacteria such as Lactobacillus, Lactococcus, etc. at high yield and titers from glucose and sucrose at temperatures between 30° C. and 40° C. (8, 10). Derivatives of Escherichia coli are the only known commercial D(−)-lactic acid producers (11, 12) and these also operate optimally at 40° C. or lower.
Alternative sources of fermentable sugars such as lignocellulosic biomass and improved microbial biocatalysts are needed to eliminate the use of food carbohydrates (glucose, sucrose) for lactic acid production (13). With cellulose as a feedstock, however, commercial fungal cellulases represent a significant process cost. This cost could be reduced by the development of thermotolerant biocatalysts that effectively ferment under conditions that are optimal (pH 5.0, 50° C.) for fungal cellulases (14, 15).
Lactic acid biocatalysts used by industry metabolize pentose sugars (from hemicellulose) by the phosphoketolase pathway, preventing efficient conversion of these sugars to lactic acid. Lactic acid produced from pentoses using this pathway is contaminated with an equimolar amount of acetic acid (FIG. 1) (8, 16-18). Attempts to improve the xylose fermentation properties of these lactic acid bacteria have met with limited success (18, 19). Although all the pentoses in hemicellulose are efficiently fermented by the E. coli derivatives to D(−)- or L(+)-lactic acid through the pentose-phosphate pathway, the temperature or pH tolerance of this biocatalyst is insufficient to permit cellulosic fermentations at the optimal temperature (50° C.) or pH (5.0) for commercial cellulases (20).
Bacillus coagulans has many desirable properties for the fermentation of lignocellulosic sugars into lactic acid. This bacterium ferments both hexoses and pentoses to L(+)-lactic acid using the highly efficient pentose phosphate pathway (FIG. 1) at 50-55° C. and pH 5.0, conditions that are optimal for commercial fungal cellulases (15, 17, 21). Native strains produce optically pure L(+)-lactic acid at concentrations as high as 180 g L−1 in fed-batch fermentations from either glucose or xylose, and perform well during simultaneous saccharification and fermentation (SSF) of cellulose using fungal cellulases (21, 22). This match in optima both for the fermentation of B. coagulans and for fungal cellulase activity allowed a 4-fold reduction in cellulase usage in comparison to SSF with mesophilic bacterial biocatalysts (21). B. coagulans also grows and ferments sugars in mineral salt medium with inexpensive corn steep liquor (0.25%, w/v) supplementation in contrast to lactic acid bacteria that require complex nutrients (8, 15, 18). Although B. coagulans has excellent potential as a biocatalyst for the conversion of cellulose to optically pure L(+)-lactic acid, an equivalent microbe for production of D(−)-isomer of lactic acid has not been previously described.